Development of a tannic acid cross-linking process for obtaining 3D porous cell-laden collagen structure

Lee, JiUn,Yeo, Miji,Kim, WonJin,Koo, YoungWon,Kim, Geun Hyung Elsevier 2018 INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES Vol.110 No.-

<P><B>Abstract</B></P> <P>Cell-printing is an emerging technique that enables to build a customized structure using biomaterials and living cells for various biomedical applications. In many biomaterials, alginate has been widely used for rapid gelation, low cost, and relatively high processability. However, biocompatibilities enhancing cell adhesion and proliferation were limited, so that, to overcome this problem, an outstanding alternative, collagen, has been extensively investigated. Many factors remain to be proven for cell-printing applications, such as printability, physical sustainability after printing, and applicability of <I>in vitro</I> cell culture. This study proposes a cell-laden collagen scaffold fabricated <I>via</I> cell-printing and tannic acid (TA) crosslinking process. The effects of the crosslinking agent (0–3wt% TA) in the cell-laden collagen scaffolds on physical properties and cellular activities using preosteoblasts (MC3T3-E1) were presented. Compared to the cell-laden collagen scaffold without TA crosslinking, the scaffold with TA crosslinking was significantly enhanced in mechanical properties, while reasonable cellular activities were observed. Concisely, this study introduces the possibility of a cell-printing process using collagen and TA crosslinking and <I>in vitro</I> cell culture for tissue regeneration.</P>

Cell-laden 3D bioprinting hydrogel matrix depending on different compositions for soft tissue engineering: Characterization and evaluation

Park, Jisun,Lee, Sang Jin,Chung, Solchan,Lee, Jun Hee,Kim, Wan Doo,Lee, Jae Young,Park, Su A Elsevier 2017 Materials Science and Engineering C Vol.71 No.-

<P>Cell-printing techniques that can construct three-dimensional (3D) structures with biocompatible materials and cells are of great interest for various biomedical applications, such as tissue engineering and drug-screening studies. For successful cell-printing with cells, bioinks are critical for both the processability of printing and the viability of printed cells. However, the influence of composition on 3D bio-printing with cells has not been well explored. In this study, we investigated different compositions of alginate bioinks by varying the concentrations of high molecular weight alginate (High Alg) and low molecular weight alginate (Low Mg). Bioinks of 3 wt% alginate containing High Mg alone or a 1:2 (Low Alg:High Alg).composite allowed for the construction of 3D scaffolds with good processability and shapes. Cell-printing with fibroblasts and in vitro culture studies revealed good viability and growth of the printed cells after up to 7 days of culture. Bioinks prepared with High and Low Mg at a 2:1 ratio exhibited better cell growth compared with those of other compositions. This study progresses the design and applications of alginate-based bioinks for cell-printing platforms in soft tissue engineering. (C) 2016 Published by Elsevier B.V.</P>

An innovative cell-printed microscale collagen model for mimicking intestinal villus epithelium

Kim, WonJin,Kim, Geun Hyung Elsevier 2018 Chemical Engineering Journal Vol.334 No.-

<P><B>Abstract</B></P> <P>Cell-printing technology for obtaining a cell-laden structure has been extensively used in tissue engineering applications due to its advantages over the conventional scaffold, which is not simultaneously fabricated with cells. To date, a realistic villi model using cell-laden bioink with both the biocompatibility and mechanical strength to achieve villus structure has not been developed. Here, we developed a human intestinal villi model with an innovative cell-printing process. The cell-laden villus structure was fabricated using a cell-laden collagen bioink cross-linked with a natural polyphenol (tannic acid). The fabricating condition was optimized and a Caco-2-laden collagen villus structure was fabricated. Using the processing conditions, a 3D collagen villus structure with appropriate geometry and a high initial cell-viability (over 90%) was obtained. <I>In vitro</I> cellular activities of the cell-laden villus structure demonstrated satisfactory cell viability with a growth-rate meaningfully higher than that in the fabricated villi structure cultured with the cell-seeding method (control). Moreover, expression of MUC17, junction marker (E-Cadherin), and alkaline phosphatase, as differentiation indicators of the epithelial cells, was significantly higher and earlier in the cell-laden structure compared to that in the control. These results indicate that the modified cell-printing process using collagen-bioink would be a highly efficient model mimicking the human intestinal epithelium.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An innovative cell-printing was developed to obtain human intestinal villus model. </LI> <LI> By optimizing processing conditions, a Caco-2-laden collagen villi was fabricated. </LI> <LI> The 3D villus model showed outstanding <I>in vitro</I> cellular activities. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Strategy to Achieve Highly Porous/Biocompatible Macroscale Cell Blocks, Using a Collagen/Genipin-bioink and an Optimal 3D Printing Process

Kim, Yong Bok,Lee, Hyeongjin,Kim, Geun Hyung American Chemical Society 2016 ACS APPLIED MATERIALS & INTERFACES Vol.8 No.47

<P>Recently, a three-dimensional (3D) bioprinting process for obtaining a cell-laden structure has been widely applied because of its ability to fabricate biomimetic complex structures embedded with and without cells. To successfully obtain a cell-laden porous block, the cell delivering vehicle, bioink, is one of the significant factors. Until now, various biocompatible hydrogels (synthetic and natural biopolymers) have been utilized in the cell-printing process, but a bioink satisfying both biocompatibility and print-ability requirements to achieve a porous structure with reasonable mechanical strength has not been issued. Here, we propose a printing strategy with optimal conditions including a safe cross-linking procedure for obtaining a 3D porous cell block composed of a biocompatible collagen-bioink and genipin, a cross-linking agent. To obtain the optimal processing conditions, we modified the 3D printing machine and selected an optimal cross-linking condition (similar to 1 mM and 1 h) of genipin solution. To show the feasibility of the process, 3D pore-interconnected cell-laden constructs were manufactured using osteoblast-like cells (MG63) and human adipose stem cells (hASCs). Under these processing conditions, a macroscale 3D collagen-based cell block of 21 X 21 x 12 mm(3) and over 95% cell viability was obtained. In vitro biological testing of the cell-laden 3D porous structure showed that the embedded cells were sufficiently viable, and their proliferation was significantly higher; the cells also exhibited increased osteogenic activities compared to the conventional alginate-based bioink (control). The results indicated the fabrication process using the collagen-bioink would be an innovative platform to design highly biocompatible and mechanically stable cell blocks.</P>

Arbitrary, Complex Cell Patterning via Inkjet Printing of a Cell Membrane-Anchoring Polymer

윤화인,조용우,최지숙,이병국,오제훈 한국고분자학회 2012 Macromolecular Research Vol.20 No.5

Well-defined cell patterns, including both adherent and non-adherent cells, were created using piezoelectric inkjet printing of a cell membrane-anchoring polymer composed of a hydrophobic oleyl group, a hydrophilic poly(ethylene glycol) (PEG) chain, and an amino-reactive N-hydroxy-succinimide (NHS) end group. Various complex living cell patterns were created by surface engineering in which inkjet-printed, membrane-anchoring polymer patterns controlled the geometric distributions of immobilized cells. Non-adherent (SNU-620) and adherent cells (HeLa) were stably anchored and patterned on a glass substrate through interactions between cell membranes and the hydrophobic oleyl groups. The effects of an apoptosis inducer (staurosporine) and an anticancer drug (paclitaxel)were visualized on cells immobilized on the polymer-printed patterns. This approach may have broad utility in most advanced biomedical devices requiring miniaturized and precisely controlled living cell patterns.

줄기세포 탑재 3차원 프린팅 polycarprolactone 스캐폴드

홍규식(Gyusik Hong),조정환(Jeong Hwan Cho),윤석환(Seokhwan Yun),최은정(Eunjeong Choi),안성민(Seongmin An),김정석(Jung Seok Kim),이재삼(Jae Sam Lee),심진형(Jin-hyung Shim),진송완(Songwan Jin),윤원수(Won-Soo Yun) 한국산학기술학회 2019 한국산학기술학회논문지 Vol.20 No.8

줄기세포를 기반으로 한 세포치료제는 생체 이식시 생착률이 낮아서 치료효과를 기대하기 어렵다. 이를 극복하기 위하여 줄기세포를 탑재할 수 있는 다양한 세포담체들이 개발되어 활용되고 있다. 이렇게 개발된 세포담체를 3-dimentional (3D) 프린팅하여 스캐폴드를 만들 경우, 환자의 손상부위 맞춤형 이식재를 제작할 수 있을 뿐만 아니라, 줄기세포를 탑재하여 손상부위를 기계적으로 보완하는 동시에 세포치료제로서의 효과도 얻을 수 있다. Polycaprolactone (PCL)은 저렴할 뿐 아니라 현재 가장 널리 쓰이고 있는 3D 프린팅 소재이기 때문에, PCL을 프린팅하여 세포담체로 활용할 경우 빠르고 경제적인 기술발전을 도모할 수 있다. 하지만 PCL 소재는 세포담체로서의 성능이 우수하지 못하여, 극히 일부의 세포만이 PCL 표면에서 생존한다. 본 연구에서는 이를 극복하기 위해서 PCL 소재에 세포의 탑재능력을 극대화되는 조건을 찾고자 하였다. PCL의 표면에 플라즈마를 처리하는 조건, PCL 표면을 콜라겐 코팅처리, PCL의 3D 프린팅 형상, 세포배양방법 변경 등 다양한 조건을 바탕으로 하여 PCL 소재에 인간 중간엽줄기세포의 세포탑재능력을 확인하였다. 세포탑재능력을 향상시킨다고 알려진 콜라겐 코팅과 플라즈마 처리를 적용하여, 플라즈마 처리 후 3% 콜라겐 코팅을 하였을 때 세포탑재능력이 가장 우수함을 확인하였고, 세포탑재능력에 영향을 줄 수 있는 세포배양방법과 스캐폴드의 구조변화를 적용하여, spheroid 세포배양시 기존의 단일세포배양법보다 탑재능력이 우수함을 확인하였으며, 스캐폴드의 구조는 세포탑재능력에 영향을 주지 못함을 확인하였다. 이를 바탕으로 PCL 소재를 세포담체로 활용한 다양한 연구를 시도하고자 한다. Stem cell therapy is not expected to bestow any therapeutic benefit because of the low engraftment rates after transplantation. Various cell-carrying scaffolds have been developed in order to overcome this problem. When the scaffold is formed by 3-dimensional (3D) printing, it is possible to create various shapes of scaffolds for specific regions of injury. At the same time, scaffolds provide stem cells as therapeutic-agents and mechanically support an injured region. PCL is not only cost effective, but it is also a widely used material for 3D printing. Therefore, rapid and economical technology development can be achieved when PCL is printed and used as a cell carrier. Yet PCL materials do not perform well as cell carriers, and only a few cells survive on the PCL surface. In this study, we tried to determine the conditions that maximize the cell-loading capacity on the PCL surface to overcome this issue. By applying a plasma treated condition and then collagen coating known to improve the cell loading capacity, it was confirmed that the 3% collagen coating after plasma treatment showed the best cell engraftment capacity during 72 hours after cell loading. By applying the spheroid cell culture method and scaffold structure change, which can affect the cell loading ability, the spheroid cell culture methods vastly improved cell engraftment, and the scaffold structure did not affect the cell engraftment properties. We will conduct further experiments using PCL material as a cell carrier and as based the excellent results of this study.

3D cell printing of <i>in vitro</i> stabilized skin model and <i>in vivo</i> pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering

Kim, Byoung Soo,Kwon, Yang Woo,Kong, Jeong-Sik,Park, Gyu Tae,Gao, Ge,Han, Wonil,Kim, Moon-Bum,Lee, Hyungseok,Kim, Jae Ho,Cho, Dong-Woo Elsevier 2018 Biomaterials Vol.168 No.-

<P><B>Abstract</B></P> <P>3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during <I>in vitro</I> tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote <I>in vivo</I> wound healing. <I>In vivo</I> results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing.</P>

Multifunctional Cell-Culture Platform for Aligned Cell Sheet Monitoring, Transfer Printing, and Therapy

Kim, Seok Joo,Cho, Hye Rim,Cho, Kyoung Won,Qiao, Shutao,Rhim, Jung Soo,Soh, Min,Kim, Taeho,Choi, Moon Kee,Choi, Changsoon,Park, Inhyuk,Hwang, Nathaniel S.,Hyeon, Taeghwan,Choi, Seung Hong,Lu, Nanshu,K American Chemical Society 2015 ACS NANO Vol.9 No.3

<P>While several functional platforms for cell culturing have been proposed for cell sheet engineering, a soft integrated system enabling <I>in vitro</I> physiological monitoring of aligned cells prior to their <I>in vivo</I> applications in tissue regeneration has not been reported. Here, we present a multifunctional, soft cell-culture platform equipped with ultrathin stretchable nanomembrane sensors and graphene-nanoribbon cell aligners, whose system modulus is matched with target tissues. This multifunctional platform is capable of aligning plated cells and <I>in situ</I> monitoring of cellular physiological characteristics during proliferation and differentiation. In addition, it is successfully applied as an <I>in vitro</I> muscle-on-a-chip testing platform. Finally, a simple but high-yield transfer printing mechanism is proposed to deliver cell sheets for scaffold-free, localized cell therapy <I>in vivo</I>. The muscle-mimicking stiffness of the platform allows the high-yield transfer printing of multiple cell sheets and results in successful therapies in diseased animal models. Expansion of current results to stem cells will provide unique opportunities for emerging classes of tissue engineering and cell therapy technologies.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancac3/2015/ancac3.2015.9.issue-3/nn5064634/production/images/medium/nn-2014-064634_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/nn5064634'>ACS Electronic Supporting Info</A></P>

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets

Ryu, Seungmi,Yoo, Jin,Jang, Yeongseon,Han, Jin,Yu, Seung Jung,Park, Jooyeon,Jung, Seon Yeop,Ahn, Kyung Hyun,Im, Sung Gap,Char, Kookheon,Kim, Byung-Soo American Chemical Society 2015 ACS NANO Vol.9 No.10

<P>Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancac3/2015/ancac3.2015.9.issue-10/acsnano.5b03823/production/images/medium/nn-2015-038237_0012.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/nn5b03823'>ACS Electronic Supporting Info</A></P>

High Aspect Ratio Conductive Lines Fabricated by Electrohydrodynamic (EHD) Jet Printing

Ayodya Pradhipta Tenggara,Yonghee Jang,Hyowon Tak,Hadi Teguh Yudistira,Vu Dat Nguyen,Doyoung Byun 대한기계학회 2014 대한기계학회 춘추학술대회 Vol.2014 No.11

Fabricating high-density integrated circuits require three dimensional electrode lines with high aspect-ratio. Nowadays, many electrode lines for electronic circuits are fabricated by printing technologies to give simple and environmental friendly fabrication technique. However, most applications of printing technologies are in two dimensional structures. In this study, we propose printing technology to fabricate three dimensional electrode lines. Electrohydrodynamic (EHD) jet printing with printing speed up to 800 mm/s was used to make three dimensional electrode lines with aspect ratio greater than 5 by using multiple printing methods. Two kinds of NPK silver inks with viscosity 5000 and 8000 cP were used. Parametric studies were done to observe the effect of printing parameters, i.e. printing numbers and printing speed into the aspect ratio of the lines. The results showed that increasing printing numbers and printing speed could increase the aspect ratio of lines. The high aspect ratio lines conducted by this study have great potential to improve efficiency of solar cells.